Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. Previously we developed a cell culture system that permitted HEV replication and produced infectious virions but these virions were retained within the cells and could be liberated only by lysing the cells. In FY2009, in order to study a more complete replication cycle of HEV, we developed a unique cell-culture system in which wild-type genomes replicated and produced infectious virus particles that were released into the culture medium in the absence of cell lysis. We used the previous cell line and the new cell line to perform comparative studies of wild-type virus and various mutants we constructed. These studies permitted us to identify the viral protein that is responsible for virus egress. Cellular proteins and components which interact with this viral protein are being identified in order to define the pathway for virus release. In FY2009 we continued our studies of genetic diversity and its implications for zoonotic spread of the virus. Egypt is unusual in that the Egyptian population has a very high seroprevalence of anti-HEV but has comparatively little hepatitis E disease, suggesting a relatively avirulent strain might be circulating there. In FY2009, two virus strains isolated in Egypt from two hepatitis E patients were amplified and sequenced to identify their genotype. Both were genotype 1, one of the two genotypes limited solely to humans. In 1997 we had discovered that swine were zoonotic hosts for genotype 3 HEV. In FY2009 we carried out studies to determine if goats, another common food source for humans, might be an additional zoonotic reservoir. Goats inoculated with 3 well-characterized infectious stocks of 3 of the 4 known human genotypes of HEV did not become infected. However, we did discover naturally-acquired antibody that reacts with human HEV capsid protein in goats being raised on nearby farms. Therefore, we performed a prospective study of goats from one of these farms. The results from that study are still being evaluated. It is not known whether receptor specificity or some other factor determines whether genotypes 3 and 4 are able to infect both primates and swine whereas genotypes 1 and 2 are restricted to primates. However, in vitro studies we performed in FY2009, suggested that barriers to virus replication within a non-primate cell exist and may play a role in host-range restriction of genotype 1 HEV.